The invention relates to erythropoiein analog-human serum albumin (EPOa-hSA) fusion proteins, nucleic acids which encode EPOa-hSA fusion proteins, and methods of making and using EPOa-hSA fusion proteins and nucleic acids.
In general, the invention features, an EPOa-hSA fusion protein, wherein at least one amino acid residue of the EPOa moiety of the fusion protein is altered such that a site which serves as a site for glycosylation in erythropoietin (EPO) does not serve as a site for glycosylation in the EPOa, e.g., an EPOa-hSA fusion protein in which at least one amino acid residue which can serve as a glycosylation site in erythropoietin is altered, e.g., by substitution or deletion, such that it does not serve as a glycosylation site.
In a preferred embodiment, the EPOa-hSA fusion protein has the formula: R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein R1 is an EPOa amino acid sequence, L is a peptide linker and R2 is human serum albumin amino acid sequence. Preferably, R1 and R2 are covalently linked via the peptide linker.
In a preferred embodiment: an amino acid residue of EPO which serves as an attachment point for glycosylation has been deleted; an amino acid residue of EPO which serves as a site for glycosylation has been replaced with an amino acid residue which does not serve as a site for glycosylation; the amino acid residue which is altered is selected from the group consisting of amino acid residues Asn24, Asn38, Asn83 and Ser126; the glycosylation site at amino acid residue Ser126 and at least one additional N-linked glycosylation site selected from the group consisting of Asn24, Asn38 and Asn83 are altered; a glycosylation site which provides for N-linked glycosylation is altered by replacing an Asn residue with an amino acid residue other than it, e.g., Gln; a glycosylation site which provides for O-linked glycosylation is altered by replacing a Ser residue with an amino acid residue other than it, e.g., Ala.
In preferred embodiments, the EPOa-hSA fusion protein is made in a mammary gland of a transgenic mammal, e.g., a ruminant, e.g., a goat.
In preferred embodiments, the EPOa-hSA fusion protein is secreted into the milk of a transgenic mammal, e.g., a ruminant, e.g., a goat.
In preferred embodiments, the EPOa-hSA fusion protein is made, in a transgenic animal, under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter. Preferably, the promoter is a goat xcex2 casein promoter.
In preferred embodiments, the EPOa-hSA fusion protein, in a transgenic animal, and is secreted into the milk of a transgenic mammal at concentrations of at least about 0.2 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml or higher.
In a preferred embodiment, amino acid residue Asn24 has been altered, e.g., substituted or deleted. Preferably, the amino acid residue Asn24 has been replaced with Gln.
In a preferred embodiment, amino acid residue Asn38 has been altered, e.g., substituted or deleted. Preferably, amino acid residue Asn38 has been replaced with Gln.
In a preferred embodiment, amino acid residue Asn83 has been altered, e.g., substituted or deleted. Preferably, the amino acid residue Asn83 has been replaced with Gln.
In yet another embodiment, amino acid residue Ser126 has been altered, e.g., substituted or deleted. Preferably, the amino acid residue Ser126 has been replaced with Ala.
In a preferred embodiment: each of amino acid residue Asn24, Asn38, Asn83 and Ser126 has been altered, e.g., substituted or deleted, such that it does not serve as a glycosylation site; each of the amino acid residues Asn24, Asn28, Asn83 and Ser126 has, respectively, been replaced with Gln, Gln, Gln, and Ala.
In a preferred embodiment, the fusion protein includes a peptide linker and the peptide linker has one or more of the following characteristics: a) it allows for the rotation of the erythropoietin analog amino acid sequence and the human serum albumin amino acid sequence relative to each other; b) it is resistant to digestion by proteases; and c) it does not interact with the erythropoietin analog or the human serum albumin.
In a preferred embodiment: the fusion protein includes a peptide linker and the peptide linker is 5 to 60, more preferably, 10 to 30, amino acids in length; the peptide linker is 20 amino acids in length; the peptide linker is 17 amino acids in length; each of the amino acids in the peptide linker is selected from the group consisting of Gly, Ser, Asn, Thr and Ala; the peptide linker includes a Gly-Ser element.
In a preferred embodiment, the fusion protein includes a peptide linker and the peptide linker includes a sequence having the formula (Ser-Gly-Gly-Gly-Gly)y wherein y is 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, the peptide linker includes a sequence having the formula (Ser-Gly-Gly-Gly-Gly)3. Preferably, the peptide linker includes a sequence having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro).
In a preferred embodiment, the fusion protein includes a peptide linker and the peptide linker includes a sequence having the formula (Ser-Ser-Ser-Ser-Gly)y wherein y is 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, the peptide linker includes a sequence having the formula ((Ser-Ser-Ser-Ser-Gly)3-Ser-Pro).
In another aspect, the invention features, an EPOa-hSA fusion protein wherein the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO (i.e., only amino acids 24, 38, 83, and 126 differ from wild type).
In another aspect, the invention features, an EPOa-hSA fusion protein which includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In another aspect, the invention features, an EPOa-hSA fusion protein which includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, an isolated nucleic acid having a nucleotide sequence which encodes an EPOa-hSA fusion protein described herein, e.g., an EPOa-hSA fusion protein wherein at least one amino acid residue is altered such that a site which serves as a site for glycosylation in EPO does not serve as a site for glycosylation in the EPOa, e.g., an EPOa-hSA fusion protein in which at least one amino acid residue of the encoded EPOa-hSA which can serve as a glycosylation site in erythropoietin is altered, e.g., by substitution or deletion, such that it does not serve as a glycosylation site.
In another aspect, the invention features, a nucleic acid which encodes an EPOa-hSA fusion protein wherein the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a nucleic acid which encodes an EPOa-hSA fusion protein which includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In another aspect, the invention features, a nucleic acid which encodes an EPOa-hSA fusion protein which includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, an expression vector or a construct which includes a nucleic acid of the invention.
In a preferred embodiment, the vector or construct further includes: a promoter; a selectable marker; an origin of replication; or a DNA homologous to a species other than human, e.g., goat DNA.
In preferred embodiments, the promoter is a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter is a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter. Preferably, the promoter is a goat xcex2 casein promoter.
In another aspect, the invention features, a cell which includes a vector or nucleic acid of the invention.
In another aspect, the invention features, a method of making an EPOa-hSA fusion in a nucleic acid construct or a vector. The method includes, forming in the construct or vector, a sequence in which a nucleic acid which encodes an erythropoietin analog is linked in frame to a nucleic acid which encodes human serum albumin.
In another aspect, the invention features, a method for making an EPOa-hSA fusion protein, e.g., from a cultured cell. The method includes supplying a cell which includes a nucleic acid which encodes an EPOa-hSA fusion protein, and expressing the EPOa-hSA fusion protein from the nucleic acid, thereby making the EPOa-hSA fusion protein.
In a preferred embodiment, the cell is a mammalian, yeast, plant, insect, or bacterial cell. Suitable mammalian cells include CHO cells or other similar expression systems.
In a preferred embodiment, the cell is a microbial cell, a cultured cell, or a cell from a cell line.
In a preferred embodiment, the EPOa-hSA fusion protein is released into culture medium.
In a preferred embodiment, the EPOa-hSA is released into culture medium and the method further includes purifying the EPOa-hSA fusion protein from culture medium.
In a preferred embodiment, the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
The invention also includes a cultured cell which includes a nucleic acid which encodes an EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion protein described herein. The invention also includes methods of making such cells, e.g., by introducing into the cell, or forming in the cell, a nucleic acid which encodes an EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion protein described herein.
In another aspect, the invention features, a method of making an EPOa-hSA fusion protein, e.g., an EPOa-hSA described herein. The method includes providing a transgenic organism which includes a transgene which directs the expression of EPOa-hSA fusion protein; allowing the transgene to be expressed; and, preferably, recovering a transgenically produced EPOa-hSA fusion protein, e.g., from the organism or from a product produced by the organism.
In a preferred embodiment, the transgenic organism is a transgenic animal, e.g., a transgenic mammal, e.g., a transgenic dairy animal, e.g., a transgenic goat or a transgenic cow.
In a preferred embodiment, the EPOa-hSA fusion protein is secreted into a bodily fluid and the method further includes purifying the EPOa-hSA fusion protein from the bodily fluid.
In a preferred embodiment, the transgenically produced EPOa-hSA fusion protein is made in a mammary gland of a transgenic mammal, preferably under the control of a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter. Preferably, the promoter is a goat xcex2 casein promoter.
In preferred embodiments, the EPOa-hSA fusion protein is made in a mammary gland of the transgenic mammal, e.g., a ruminant, e.g., a dairy animal, e.g., a goat or cow.
In preferred embodiments, the EPOa-hSA fusion protein is secreted into the milk of a transgenic mammal at concentrations of at least about 0.2 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml or higher.
In preferred embodiments the method further includes recovering EPOa-hSA fusion protein from the organism or from a product produced by the organism, e.g., milk, seeds, hair, blood, eggs, or urine.
In yet another embodiment, the EPOa-hSA fusion protein is produced in a transgenic plant.
In a preferred embodiment, the erythropoietin analog includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a method of making a transgenic EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion described herein. The method includes providing a transgenic animal, e.g., goat or a cow, which includes a transgene which provides for the expression of the EPOa-hSA fusion protein; allowing the transgene to be expressed; and, preferably, recovering EPOa-hSA fusion protein, from the milk of the transgenic animal.
In preferred embodiments, the EPOa-hSA fusion protein is made in a mammary gland of the transgenic mammal, e.g., a ruminant, e.g., a goat or a cow.
In preferred embodiments, the EPOa-hSA fusion protein is secreted into the milk of the transgenic mammal, e.g., a ruminant, e.g., a dairy animal, e.g., a goat or a cow.
In preferred embodiments, the EPOa-hSA fusion protein is made under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter. Preferably, the promoter is a goat xcex2 casein promoter.
In preferred embodiments, the EPOa-hSA fusion protein is secreted into the milk of a transgenic mammal at concentrations of at least about 0.2 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml or higher.
In a preferred embodiment, the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a method for providing a transgenic preparation which includes an EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion protein described herein, in the milk of a transgenic mammal. The method includes: providing a transgenic mammal having an EPOa-hSA fusion protein-coding sequence operatively linked to a promoter sequence that results in the expression of the protein-coding sequence in mammary gland epithelial cells, allowing the fusion protein to be expressed, and obtaining milk from the mammal, thereby providing the transgenic preparation.
In a preferred embodiment, the EPOa-hSA fusion protein-coding sequence operatively linked to a promoter sequence is introduced into the germline of the transgenic mammal.
In a preferred embodiment, the erythropoietin analog includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Alal 26 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serun albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a method for providing a transgenic preparation which includes an EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion protein described herein, in the milk of a transgenic goat or transgenic cow. The method includes providing a transgenic goat or cow having an EPOa-hSA fusion protein-coding sequence operatively linked to a promoter sequence that results in the expression of the protein-coding sequence in mammary gland epithelial cells, allowing the fusion protein to be expressed, and obtaining milk from the goat or cow, thereby providing the transgenic preparation.
In a preferred embodiment, the erythropoietin analog includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a transgenic organism, which includes a transgene which encodes an EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion protein described herein.
In a preferred embodiment, the transgenic organism is a transgenic plant or animal. Preferred transgenic animals include: mammals; birds; reptiles; marsupials; and amphibians. Suitable mammals include: ruminants; ungulates; domesticated mammals; and dairy animals. Particularly preferred animals include: mice, goats, sheep, camels, rabbits, cows, pigs, horses, oxen, and llamas. Suitable birds include chickens, geese, and turkeys. Where the transgenic protein is secreted into the milk of a transgenic animal, the animal should be able to produce at least 1, and more preferably at least 10, or 100, liters of milk per year.
In preferred embodiments, the EPOa-hSA fusion protein is under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter. Preferably, the promoter is a goat xcex2 casein promoter.
In a preferred embodiments, the EPOa-hSA fusion protein is secreted into the milk at concentrations of at least about 0.2 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml or higher.
In a preferred embodiment, the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln4, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala26, a peptide linker, e.g. , a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln124, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a transgenic cow, goat or sheep, which includes a transgene which encodes an EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion protein described herein.
In preferred embodiments, the EPOa-hSA fusion protein is under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter. Preferably, the promoter is a goat xcex2 casein promoter.
In preferred embodiments, the EPOa-hSA fusion protein is secreted into the milk at concentrations of at least about 0.2 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml or higher.
In a preferred embodiment, the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a herd of transgenic animals having at least one female and one male transgenic animal, wherein each animal includes an EPOa-hSA fusion protein transgene, e.g., a transgene which encodes an EPOa-hSA fusion protein described herein.
In a preferred embodiment, a transgenic animal of the herd is a mammal, bird, reptile, marsupial or amphibian. Suitable mammals include: ruminants; ungulates; domesticated mammals; and dairy animals. Particularly preferred animals include: mice, goats, sheep, camels, rabbits, cows, pigs, horses, oxen, and llamas. Suitable birds include chickens, geese, and turkeys. Where the transgenic protein is secreted into the milk of a transgenic animal, the animal should be able to produce at least 1, and more preferably at least 10, or 100, liters of milk per year.
In preferred embodiments, the EPOa-hSA fusion protein is under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can is a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter.
In preferred embodiments, the EPOa-hSA fusion protein is secreted into the milk at concentrations of at least about 0.2 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml or higher.
In a preferred embodiment, the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a pharmaceutical composition having a therapeutically effective amount of an EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion protein described herein, and a pharmaceutically acceptable carrier.
In a preferred embodiment, the composition includes milk.
In a preferred embodiment, the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a kit having an EPOa-hSA fusion protein, e.g., an EPOa-hSA fusion protein described herein, packaged with instructions for treating a subject in need of eiythropoietin.
In a preferred embodiment, the subject is a patient suffering from anemia associated with renal failure, chronic disease, HIV infection, blood loss or cancer.
In another preferred embodiment, the subject is a preoperative patient.
In a preferred embodiment, the erythropoietin analog includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a purified preparation of an EPOa-hSA fusion protein, e.g., an EPO-hSA fusion protein described herein.
In preferred embodiments, the preparation includes at least 1, 10, 100 or 1000 micrograms of EPOa-hSA fusion protein. In preferred embodiments, the preparation includes at least 1, 10, 100 or 1000 milligrams of EPOa-hSA fusion protein.
In another aspect, the invention features, an EPOa-hSA fusion protein, or a purified preparation thereof, wherein the erythropoietin analog includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In preferred embodiments, the preparation includes at least 1, 10, 100 or 1000 micrograms of EPOa-hSA fusion protein. In preferred embodiments, the preparation includes at least 1, 10, 100 or 1000 milligrams of EPOa-hSA fusion protein.
In another aspect, the invention features, an EPOa-hSA fusion protein, or a purified preparation thereof, which includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In preferred embodiments, the preparation includes at least 1, 10, 100 or 1000 micrograms of EPOa-hSA fusion protein. In preferred embodiments, the preparation includes at least 1, 10, 100 or 1000 milligrams of EPOa-hSA fusion protein.
In another aspect, the invention features, an EPOa-hSA fusion protein, or a purified preparation thereof, which includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In preferred embodiments, the preparation includes at least 1, 10, or 100 milligrams of EPOa-hSA fusion protein. In preferred embodiments, the preparation includes at least 1, 10, or 100 grams of EPOa-hSA fusion protein.
In another aspect, the invention features, a method of treating a subject, e.g., a human, in need of erythropoietin. The method includes administering a therapeutically effective amount of an EPOa-hSA fusion protein, e.g., an EPO-hSA fusion protein described herein, to the subject.
In a preferred embodiment, the subject is a patient suffering from anemia associated with renal failure, chronic disease, HIV infection, blood loss or cancer.
In another preferred embodiment, the subject is a preoperative patient.
In preferred embodiments the EPOa-hSA is administered repeatedly, e.g., at least two, three, five, or 10 times.
In a preferred embodiment, the erythropoietin analog includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a method of treating a subject in need of erythropoietin. The method includes delivering or providing a nucleic acid encoding an EPOa-hSA fusion protein, e.g., a fusion protein described herein, to the subject.
In a preferred embodiment, the nucleic acid is delivered to a target cell of the subject.
In a preferred embodiment, the nucleic acid is delivered or provided in a biologically effective carrier, e.g., an expression vector.
In a preferred embodiment, the nucleic acid is delivered or provided in a cell, e.g., an autologous, allogeneic, or xenogeneic cell.
In a preferred embodiment, the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a method of making a transgenic organism which has an EPOa-hSA transgene. The method includes providing or forming in a cell of an organism, an EPOa-hSA transgene, e.g., a transgene which encodes an EPOa-hSA fusion protein described herein; and allowing the cell, or a descendent of the cell, to give rise to a transgenic organism.
In a preferred embodiment, the transgenic organism is a transgenic plant or animal. Preferred transgenic animals include: mammals; birds; reptiles; marsupials; and amphibians. Suitable mammals include: ruminants; ungulates; domesticated mammals; and dairy animals. Particularly preferred animals include: mice, goats, sheep, camels, rabbits, cows, pigs, horses, oxen, and llamas. Suitable birds include chickens, geese, and turkeys. Where the transgenic protein is secreted into the milk of a transgenic animal, the animal should be able to produce at least 1, and more preferably at least 10, or 100, liters of milk per year.
In preferred embodiments, the EPOa-hSA fusion protein is under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter. Preferably, the promoter is a goat xcex2 casein promoter.
In preferred embodiments, the organism is a mammal, and the EPOa-hSA fusion protein is secreted into the milk of the transgenic animal at concentrations of at least about 0.2 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml or higher.
In a preferred embodiment, the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment, the EPOa-hSA fusion protein includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and human serum albumin.
In a preferred embodiment, the EPOa-hSA fusion protein includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)3-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, an erythropoietin analog (EPOa) protein, or a purified preparation thereof, e.g., the EPOa moiety of an EPOa-hSA fusion protein described herein, wherein at least one amino acid residue is altered such that a site which serves as a site for glycosylation in EPO, does not serve as a site for glycosylation in the EPOa, e.g., an EPOa in which at least one amino acid residue which can serve as a glycosylation site in erythropoietin is altered, e.g., by substitution or deletion, such that it does not serve as a glycosylation site.
In a preferred embodiment, the erythropoietin analog includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, an isolated nucleic acid having a nucleotide sequence which encodes an EPOa described herein.
In another aspect, the invention features, an expression vector or a construct which includes an EPOa nucleic acid described herein.
In a preferred embodiment, the vector or construct further includes: a promoter; a selectable marker; an origin of replication; or a DNA homologous to a species other than human, e.g., goat DNA.
In another aspect, the invention features, a cell which includes a vector or construct which includes an EPOa nucleic acid described herein.
A purified preparation, substantially pure preparation of a polypeptide, or an isolated polypeptide as used herein, means a polypeptide that has been separated from at least one other protein, lipid, or nucleic acid with which it occurs in the cell or organism which expresses it, e.g., from a protein, lipid, or nucleic acid in a transgenic animal or in a fluid, e.g., milk, or other substance, e.g., an egg, produced by a transgenic animal. The polypeptide is preferably separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. The polypeptide preferably constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 xcexcg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
As used herein, xe2x80x9chuman serum albuminxe2x80x9d or xe2x80x9chSAxe2x80x9d refers to a polypeptide having the amino acid sequence described in Minghetti et al. J Biol. Chem. 261:6747-6757, 1986; Lawn et al. NucL Acids Res. 9:6103, 1981. In preferred embodiments, sequence variations are included wherein one or up to two, five, 10, or 20 amino acid residues have been substituted, inserted or deleted. Variants will have substantially the same immunogenicity, in, e.g., mice, rats, rabbits, primates, baboons, or humans, as does hSA. Variants, when incorporated into a fusion protein which includes EPOa, will result in an EPOa-hSA a fusion which has similar clearance time, in e.g., mice, rabbits, or humans, and activity as does a fusion protein which includes the EPOa and hSA. As used herein, xe2x80x9cerythropoietinxe2x80x9d or xe2x80x9cEPOxe2x80x9d refers to a glycoprotein hormone involved in the maturation of erythroid progenitor cells into erythrocytes. The sequence of EPO can be found in Powell, J.S., et al., Proc. Natl. Acad. Sci. USA, 83:6465-6469 (1986).
A substantially pure nucleic acid, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a CDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional EPOa-hSA fusion protein sequence.
Homology, or sequence identity, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid xe2x80x9chomologyxe2x80x9d is equivalent to amino acid or nucleic acid xe2x80x9cidentityxe2x80x9d). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology =# of identical positions/total # of positionsxc3x97100). For example, if 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology or sequence identity.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to ITALY nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to ITALY protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The terms peptides, proteins, and polypeptides are used interchangeably herein.
As used herein, the term transgene means a nucleic acid sequence (encoding, e.g., one or more EPOa-hSA fusion protein polypeptides), which is introduced into the genome of a transgenic organism. A transgene can include one or more transcriptional regulatory sequences and other nucleic acid, such as introns, that may be necessary for optimal expression and secretion of a nucleic acid encoding the fusion protein. A transgene can include an enhancer sequence. An EPOa-hSA fusion protein sequence can be operatively linked to a tissue specific promoter, e.g., mammary gland specific promoter sequence that results in the secretion of the protein in the milk of a transgenic mammal, a urine specific promoter, or an egg specific promoter.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
A transgenic organism, as used herein, refers to a transgenic animal or plant.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is a non-human animal in which one or more, and preferably essentially all, of the cells of the animal contain a transgene introduced by way of human intervention, such as by transgenic techniques known in the art. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
As used herein, a xe2x80x9ctransgenic plantxe2x80x9d is a plant, preferably a multi-celled or higher plant, in which one or more, and preferably essentially all, of the cells of the plant contain a transgene introduced by way of human intervention, such as by transgenic techniques known in the art.
Mammals are defined herein as all animals, excluding humans, that have mammary glands and produce milk.
As used herein, a xe2x80x9cdairy animalxe2x80x9d refers to a milk producing non-human animal which is larger than a rodent. In preferred embodiments, the dairy animal produce large volumes of milk and have long lactating periods, e.g., cows or goats.
As used herein, the term xe2x80x9cplantxe2x80x9d refers to either a whole plant, a plant part, a plant cell, or a group of plant cells. The class of plants which can be used in methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. It includes plants of a variety of ploidy levels, including polyploid, diploid and haploid.
As used herein, the term xe2x80x9cformulationxe2x80x9d refers to a composition in solid, e.g., powder, or liquid form, which includes an EPOa-hSA fusion protein. Formulations can provide therapeutical or nutritional benefits. In preferred embodiments, formulations can include at least one nutritional component other than EPOa-hSA fusion protein. A formulation can contain a preservative to prevent the growth of microorganisms.
As used herein, the term xe2x80x9cnutraceutical, xe2x80x9d refers to a food substance or part of a food, which includes an EPOa-hSA fusion protein. Nutraceuticals can provide medical or health benefits, including the prevention, treatment or cure of a disorder. The transgenic protein will often be present in the nutraceutical at concentration of at least 100 mg/kg, more preferably at least 1 mg/kg, most preferably at least 10 mg/kg. A nutraceutical can include the milk of a transgenic animal.
As used herein, the term xe2x80x9cerythropoietin analogxe2x80x9d or xe2x80x9cEPOaxe2x80x9d refers to an EPO molecule which differs from a naturally occurring or recombinant EPO at one or more amino acids. Preferably, the EPO analog differs from a naturally occurring or recombinant human EPO at one or more of the following amino acids: Asn24, Asn38, Asn83 and Ser126. Unless otherwise stated, EPO and EPOa as used herein refer to human EPO and EPOa.
A polypeptide has EPOa-hSA fusion protein biological activity if it has at least one biological activity of EPO or is an antagonist, agonist, or super-agonist of a polypeptide having a biological activity of EPO.
As used herein, the language xe2x80x9csubjectxe2x80x9d includes human and non-human animals. The term xe2x80x9cnon-human animalsxe2x80x9d of the invention includes vertebrates, e.g., mammals and non-mammals, such as non-human primates, ruminants, birds, amphibians, reptiles and rodents, e.g., mice and rats. The term also includes rabbits.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.